Towards Understanding the Molecular Basis of Outer Membrane Vesicles Formation

• Author / Creator

  Elhenawy,Wael

• The release of outer membrane vesicles (OMV) is utilized by Gram-negative bacteria to perform a myriad of functions during pathogenesis and symbiosis. Despite their well-recognized roles, the mechanism responsible for OMV formation in bacteria remains elusive. Whether OMV are produced by an active mechanism or by the passive disintegration of the outer membrane (OM) is a still matter of controversy. If OMV are produced as a consequence to cell lysis, then their protein content should not differ from the parent OM. Nonetheless, many studies demonstrated the differences between the proteomes of OMV and the originating membranes, suggesting the presence of a directed mechanism for OMV formation and cargo selection.
  In this thesis, we compared the proteomes of OM and OMV purified from B. fragilis and B. thetaiotaomicron. Using mass spectrometry, we identified the proteins in the OM and OMV of both organisms. Our analysis revealed the presence of more than forty proteins exclusively-packed in B. fragilis OMV. In parallel, more than 30 proteins were excluded from OMV and were detected only in B. fragilis OM. Sugar hydrolases and proteases constituted a significant fraction of the OMV specific proteins. Using in vitro biochemical assays, we demonstrated the hydrolytic activity of B. fragilis OMV. Moreover, we were able to show that B. fragilis can induce OMV hydrolases in response to extracellular cues. Similar results were obtained with B. thetaiotaomicron OMV analysis.
  OMV cargo selection is not restricted to the protein content but was found to include the packed lipids. Previous studies demonstrated the uneven distribution of lipids between the OMV and the parent OM. In the dental pathogen, Porphyromonas gingivalis, deacylated lipid A species were preferentially enriched in the OMV. In this thesis, we investigated the role of the lipid A deacylase, PagL, in OMV production by the prominent enteric pathogen, Salmonella enterica serovar Typhimurium. The expression of PagL in vitro resulted in the exclusive accumulation of deacylated lipid A forms in S. Typhimurium OMV. Additionally, more OMV production was observed when PagL was recombinantly expressed in S. Typhimurium. On contrary, the expression of a catalytically inactive variant of the enzyme did not induce similar vesiculation levels. Moreover, we demonstrated that PagL is involved in OMV formation by intracellular S. Typhimurium. Our results suggest a role for PagL-mediated lipid A deacylation in OMV biogenesis. Unraveling the molecular mechanisms mediating OMV formation will greatly improve our understanding of bacterial pathogenesis.

• Subjects / Keywords

  • OMV cargo selection

• Graduation date

  Fall 2015

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3BR8MN0J

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.